BSA 4204

PLANT BIOTECHNOLOGY
Credit Units: 04

LECTURE 2
INTRODUCTION TO MOLECULAR BIOLOGY
-NUCLEIC ACIDS AND PCR TECHNOLOGY

BY ROBERT AMAYO
 MOLECULAR BIOLOGY
• Study of the composition, structure, and interactions of
cellular molecules (nucleic acids, proteins, and protein
derivatives), that carry out the biological processes.
• Study of the molecular basis of the biological activities
occurring in and between cells such as;
• Biological syntheses
• Biological modification
• Expression of genes
• Regulation of biological interactions

• It plays a critical role in the understanding of structures,

functions, and internal controls within individual cells.
 Key areas of molecular biology
• Polymerase Chain Reaction (PCR) technology
• Genomics
• Proteomics
• Molecular Microbiology
• Genetic transformation/engineering
• Molecular modeling
• Molecular breeding
• Molecular marker selection
• Mutation
• Bioinformatics
• Genetic fingerprinting
• Cryopreservation
THE CENTRAL DOGMA OF MOLECULAR
BIOLOGY
 KEY MOLECULAR COMPONENTS OF THE CELL

• These components are key building blocks of

biological activities in organisms.
• They include;
1) Deoxyribonucleic acid (DNA)
2) Ribonucleic acid (RNA)
i. mRNA
ii. rRNA
iii. tRNA
3) Protein/amino acids
 NUCLEIC ACIDS
• These are naturally occurring chemical compounds that are
made up of nucleotides ( made of phosphoric acid, sugars, and
a mixture of organic bases - purines and pyrimidines).
• They are primary information-carrying molecules in cells and
they make up the genetic material of an organism.
• They include;
– Deoxyribonucleic acid (DNA) – this is the master blueprint
for life and constitutes the genetic material in all free-living
organisms and some viruses.
– Ribonucleic acid (RNA) – this is the genetic material of
viruses, but it is also found in all living cells, where it plays
an important role in certain processes such as protein
biosynthesis.
BASIC STRUCTURE OF NUCLEIC ACIDS
Key terms
• Nucleobases
• Nucleotide
• Nucleoside
• Polynucleotide
• Purines
• Pyrimidines
• Ribose
• De-oxyribose
• Phosphodiester
bonds
• Precursors
• Hydrogen bonds
Biosynthesis and degradation of nucleic
acids/ nucleic acid metabolism
• Synthesis and degradation of the nucleic acids occurs in
the cell
• The synthesis is initiated during pentose phosphate
pathway (PPP) – formation of ribose phosphate portion.
• Formation of the nucleobases and nucleosides;
– Pyrimidines – synthesized starting with Uridine (Uracil) which
is the precursor for both Cytidine (cytosine) and thymidine
(thyamine)).
– Purines - synthesized starting with Adenosine which is a
precursor for Guanosine and vice versa.
 Biosynthesis and degradation of nucleic
acids
• The phosphate group is attached to the nucleosides to
form a complete nucleotide such as Uridine 5-
monophosphate (UMP) if only one phosphate group is
attached.
• The nucleic acids are formed from different nucleotides
joined together with phosphodiester bond.
• https://youtu.be/jwMSVdosFXY
• https://youtu.be/s1MoBTEcVYY
DNA AND RNA AS GENETIC MATERIAL
DNA
• This is the genetic material of most organisms, except viruses
• It is made up of sequence of nucleotides (made from 4
nucleobases – A, G, C, or T) that encode for genetic information.
• The information is organized into chromosomes and can be
passed on from generation to generation.
• Segments of the DNA make up the gene, and carry specific
information. Other parts of the DNA play regulatory role.
• In eukaryotes, the main genetic information is stored in DNA is
found in nucleus. Others are stored in organelle DNA.
• In prokaryotes, the genetic information is stored in DNA found
in cytoplasm.
• Complexity of the DNA structure as interacts with other proteins
control transcription
DNA AND RNA AS GENETIC MATERIAL
RNA
• This is the genetic material of some viruses, in other
organisms it is a transfer ‘machine’ for genetic information.
• It is made up of sequence of nucleotides (made from 4
nucleobases – A, G, C, or U) that encode for genetic
information or transcribed information.
• The transcribed information are obtained from gene, and is
translated into amino acid sequence used for building
proteins.
• In eukaryotes, the RNA are found in Cytoplasm.
• Types of RNAs
– mRNA, rRNA, tRNA, snRNA, RNAi, etc.
– Exons vs Introns
– RNA splicing and RNA editing
 POLYMERASE CHAIN REACTION (PCR)
• PCR is a common in-vitro technique used to generate
thousands, millions or billions of copies of a specific DNA
sequence.
• This is to increase amount of DNA sequence for visibility
• It mimics DNA replication processes in the cell
• It uses;
– Primers
– Template
– Polymerase enzyme e.g. Taq polymerase
– Nucleotides (dNTPs)
– Reaction buffer
– Water
– PCR machine or Thermal cycler
• It involves repeated changes in temperature in series.
• The technique is used in cloning, diagnostics, forensic
analysis, etc.
DNA template and primer pair
 BASIC STEPS OF PCR
• Preparation
– DNA extraction , quantification and purification
– Preparation of reaction mix on ice
– Optimization
• Amplification
a) Denaturation (94-96 °C; 30 – 60 sec): The reaction is heated
strongly to separate, or denature, the DNA strands.
b) Annealing (55 – 65 °C; 30 – 45 sec): Cool the reaction so the
primers can bind to their complementary sequences on the
single-stranded template DNA.
c) Extension (72 °C; 30 – 60 sec): Raise the reaction temperatures
so Taq polymerase extends the primers, synthesizing new strands
of DNA.
d) There is initial heating (activation; (94-96 °C; 1 – 2 min): ), final
extension (72 °C; 1 – 2 min) and cooling off (4 °C; ∞).
Steps a – c are repeated in 25 – 35 cycles.
• Visualization or use of products (gel)
PCR STEPS
Visualization of
PCR products
through gel
electrophoresis
 APPLICATION OF PCR IN PLANT BIOTECHNOLOGY
1) Southern and northern hybridization,
2) Used in gene clonning, thus to check whether the gene of interest
has been cloned into the vector,
3) Genetic fingerprinting; detection and identification of gene(s) of
interest in different plants and their progenies.
4) Study inheritance of specific traits or transfer of the genes
determining a particular trait from one generation to another,
5) Disease diagnosis, thus they are used in detection and
identification of plant pathogens,
6) Screening of transgenic plants; thus to check whether gene of
interest has been transferred, deleted or silenced,
7) Determine the genome of the plant through sequencing of the
DNA,
8) Determine evolutionary relationship and footprints of plant
species.
 TYPES OF PCR
1) End point PCR
– End point PCR, as the name implies, analyzes the end product of
PCR.
– The final PCR product is often visualized on a diagnostic agarose
gel to confirm product presence, size, and relative quantity.
– It most commonly used in molecular cloning, sequencing, and
genotyping.
– It’s extremely useful but is not as quantitative as other methods
of PCR.
– End point PCR, which is commonly referred to as simply PCR, is
used for an array of different molecular biology techniques and
experiments.
– End point PCR’s goal is to amplify a specific target of DNA.
TYPES OF PCR
TYPES OF PCR
 TYPES OF PCR
2) Quantitative polymerase chain reaction (qPCR) or Real-time PCR
– This is also known as real time PCR is used for measuring DNA
concentration.
– It requires the addition of a probe based fluorescent dye that intercalates
with any dsDNA and the use of a fluorometer feature built into the
thermocycler to measure that fluorescent output.
– With this fluorescent dye, the concentration of the DNA during the PCR
reaction cycles is continuously detected via a fluorescent signal. The signal
increases proportionally to the amount of product produced each cycle.
– To determine the concentration of the starting template DNA the
fluorescent signal throughout the reaction is compared to a standard curve
of amplified DNA of a known starting concentration.
– The cycle in which the unknown DNA is detected compared to the standard
curve can be used to determine the amount of starting material the
sample.
– It can also be used to quantitate RNA levels using reverse transcription-
qPCR (RT-qPCR). The first step of this process requires RNA to be converted
to cDNA using reverse transcription and the cDNA is subsequently
quantified by qPCR.
– qPCR is used in a variety of applications including gene expression profiling,
studying copy number variation, and molecular diagnostics.
TYPES OF PCR
TYPES OF PCR
 TYPES OF PCR
3) Digital droplet PCR, ddPCR
• This method provides ultrasensitive and absolute nucleotide
concentration
• It can be used to quantify DNA sequences that are rare, for example,
rare alleles or mutations.
• It also does not require a reference or standard curve, which can be
time consuming and challenging to get right.
• It uses a water-oil emulsion droplet technology that fractions the
PCR reaction sample into approximately 20,000 droplets.
• Each droplet contains the material required for PCR amplification.
• Following the PCR reactions each droplet is analyzed by a droplet
reader, which measures the fluorescence amplitude of each droplet.
• The fraction of fluorescent PCR-positive droplets is determined and
then analyzed using Poisson statistics to determine the
concentration of the original template DNA in the sample.
TYPES OF PCR
TYPES OF PCR
 TYPES OF PCR
4) Multiplex PCR
• Multiplex PCR, as the name implies, is a method in which
multiple targets can be amplified in a single PCR
experiment using multiple primers all in one PCR
reaction.
• This is an extremely useful PCR method that can help
save time and effort in the laboratory.
• There are two main categories of multiplex PCR:
a) Single template PCR reaction - one template is amplified using
several forward and reverse primer sets.
b) Multiple template PCR - multiple templates with different primer
pairs that align to the target region of each template are used in one
reaction.
TYPES OF PCR
VARIANTS/MODIFICATION OF PCR